Evidence for endoreduplication: germ cell DNA levels prior to chromatin diminution in Mesocyclops edax.

We studied the functional significance of marked differences in the DNA content of somatic cells and germ line nuclei by static Feulgen-DNA cytophotometry for several species of microcrustaceans that exhibit chromatin diminution during very early stages of embryogenesis. Mature females and males showed many gonadal nuclei with elevated amounts of DNA that persist until dispersal of this "extra" DNA throughout the cytoplasm as fragments and coalescing droplets of chromatin during anaphase of the diminution division.

Capillary ion analysis of lithium concentrations in biological fluids and tissues of Poecilia (teleost).

Capillary ion analysis (CIA) is a form of capillary electrophoresis that uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Application of this technique for detection of lithium concentrations in plasma and tissues of Poecilia was the purpose of this investigation. CIA was performed using a 75 microm ID x 60 cm length fused-silica capillary and a run electrolyte of 67.7 mg hydroxyisobutyric acid (HIBA), 52.8 mg 18-crown-6-ether and 64 microL UV-CAT-1 reagent (4-methylbenzylamine) in a volume of 100 mL water (18 (M)omega) with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: potassium, 2.98 min; calcium, 3.48 min; sodium, 3.60 min; barium (internal standard), 4.15 min and lithium, 4.26 min. Lithium and barium migration times were stable and reproducible. Correlation coefficients (r) between peak area ratios of lithium/barium for concentrations ranging from 0.1 to 2.0 mM were from 0.976 to 0.996. Coefficients of variation (CV) for lithium concentrations ranged from 4.07 to 15.71% between days and 4.38 to 7.76% within-day. Application of this methodology for determination of lithium concentrations in the plasma, brains and livers of fish dosed with lithium for 23 days are presented. CIA is applicable to analysis of lithium concentrations in biological fluids and tissues of fish.

Quantitation of absolute Pneumocystis carinii nuclear DNA content. Trophic and cystic forms isolated from infected rat lungs are haploid organisms.

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37 degrees C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G + C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carini carinii isolated from infected rat lungs are haploid organisms.

Post-zygotic modifications and intra- and inter-individual nucleolar organizing region variations in fish: report of a case involving Leporinus friderici.

Silver nitrate staining, a rapid and efficient method, has proven to be excellent for nucleolar organizing region (NOR) studies in fish. Some fish appear to have only two NOR-bearing chromosomes in their karyotype, whereas others probably have several. In the present study we analyzed the NORs of Leporinus friderici, a species that, on the basis of previous studies, has been considered as representative of species with NORs carried by a single chromosome pair. The analyses were performed by a combination of three methods, i.e. silver nitrate staining, staining with the GC-specific fluorochrome chromomycin A3, and in situ hybridization with digoxigenin-labeled probes. The results showed that, although more frequent and conspicuous in a single chromosome pair, the NORs of this species are present in multiple chromosomes. Intra- and inter-individual variations observed by the three methods strongly suggest the occurrence of post-zygotic modifications involving NORs. NOR identification in fish, almost exclusively performed by the silver nitrate method, is currently being re-evaluated by methods such as chromomycin A3 staining and in situ hybridization, which may provide important information leading to a better understanding of chromosome evolution in these animals.

The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

Cytogenetics of bisexual/unisexual species of Poecilia. V. Unisexual poeciliids with anomalous karyotypes from northeastern Mexico.

Unisexual poeciliid fishes collected from two localities in the Soto la Marina drainage have been analyzed. Unisexual diploid and triploid specimens with anomalous karyotypes were found among karyotypically normal specimens. Chromosomal data are discussed in light of the origin of clonal diversity in unisexual/bisexual breeding complexes and with regard to the role of triploids in these breeding complexes.

Cytogenetics of bisexual/unisexual species of Poecilia. III. The karyotype of Poecilia formosa, a gynogenetic species of hybrid origin.

Chromosomes of the Amazon molly, Poecilia formosa, a unisexual species of hybrid origin, were investigated by C-banding, silver staining, and fluorescent staining with DAPI, quinacrine dihydrochloride, and chromomycin A3. Analysis of heterochromatin distribution indicates that chromosomes similar to the W chromosome of P. latipinna are not present in the unisexual species. Therefore, morphologically differentiated sex chromosomes do not form the basis of the unisexuality in P. formosa. The number and location of nucleolar organizer regions vary in P. formosa and do not correlate well with those of the parental species.

Cytogenetics of bisexual/unisexual species of Poecilia. II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining.

Chromosomes of Poecilia mexicana mexicana, one of the bisexual species involved in the hybrid origin of the unisexual teleost fish species P. formosa, were analyzed by several staining techniques. Sex-specific, differential heterochromatin, found in other congeneric species, was not observed in P. m. mexicana. Nucleolar organizer regions were polymorphic among individual specimens within a given population sample. A single specimen exhibiting intraindividual variability of chromosome pair 1 and a specimen with a triploid karyotype are also described.

Cytogenetics of bisexual/unisexual species of Poecilia. I. C-bands, Ag-NOR polymorphisms, and sex chromosomes in three populations of Poecilia latipinna.

Three populations of the North American cyprinodont fish Poecilia latipinna, considered to be one of the progenitor species of the gynogenetic unisexual P. formosa, were analyzed by C-banding and Ag-staining. C-bands were found to be polymorphic, and Ag-staining showed a high degree of variability in both the number and location of nucleolus organizer regions. The C-banding and Ag-staining patterns allow, to a certain extent, to distinguish individual specimens from each of these populations. Females of the three populations were found to have a heteromorphic chromosome pair, which was frequently identifiable with Giemsa staining and always after C-banding. This pair could be interpreted as sex chromosomes of the ZW/ZZ type.

Maturation and degeneration of the fat body in the Drosophila larva and pupa as revealed by morphometric analysis.

Using morphometric and cytochemical techniques we have described changes taking place in the fat body cells during three different stages of development. The cell number remains constant at about 2200 cells during larval life and then decreases gradually and continuously throughout metamorphosis and the first 3 days of the adult stage until no more cells can be observed. Cell size increases rapidly during the larval period and decreases steadily during metamorphosis and adult stage. The size of the nuclei increases during the larval instars and decreases during the pupal interval. The change in nuclear size is correlated with the amount of DNA present throughout development implying the nuclear DNA is synthesized during the larval period and degraded gradually during metamorphosis. The cell size changes are due in large part to accumulation or loss of reserve substances: lipid droplets, glycogen deposits and protein granules. During metamorphosis the amount of lipid decreases slightly whereas glycogen experiences two loss cycles. The protein granules in the form of lysosomes continue to increase in amount during the first day of metamorphosis because of a short period of massive autophagy. Then the lysosomes decrease in amount throughout the remainder of metamorphosis. The lysosomes stain positively for lipofuscin.

Bizarre blue nevus: a case report with deoxyribonucleic acid content analysis.

A case report of an unusual variant of blue nevus is presented. The lesion demonstrated striking cytologic atypia but lacked clinical and certain histologic features of malignancy. Similarities and differences between this lesion and atypical blue nevus and Spitz nevus are discussed. An analysis of the deoxyribonucleic acid levels in lesional cells was consistent with polyploidy. This supports the interpretation of the lesion as atypical but is not inconsistent with a benign diagnosis.

Adipose tissue of Drosophila melanogaster: VII. Distribution of nuclear DNA amounts along the anterior-posterior axis in the larval fat body.

The DNA content of fat body nuclei was measured cytophotometrically as a function of position along an anterior-posterior (A-P) axis of the tissue throughout larval development. There was a dramatic 55-fold increase in the average amount of DNA per nucleus during the first 3 days of this period, but there was no further increase during the final day. However, the rate of increase had regional specificity. The amount of DNA per nucleus correlated significantly with its position along the A-P axis of the tissue: nuclei in a posterior direction contain gradually increasing amounts of DNA. There was up to a seven-fold difference between the smallest anterior and largest posterior nucleus. In addition, for three of the ages studied there was a subgradient in the posterior region with a slope that was considerably steeper than that of the overall tissue gradient. The tissue has three characteristic morphological regions, anterior, medial, and posterior, which can be recognized early in development and which are maintained throughout the larval period. The distribution of nuclear DNA classes determined for cells in each region for the final 2 days of larval life became fixed before the final day of development. The significance of the DNA gradient in terms of a protein storage gradient is discussed.

Cytophotometric evidence of variation in genome size of desmognathine salamanders.

The amount of DNA per haploid genome, the C-value, is often directly correlated with nuclear and cell volume, but inversely correlated with cell replication rate. Also, rates of cellular growth sometimes appear to be correlated with organismal developmental rates and life history patterns. Among vertebrates, salamanders exhibit the greatest variation in genome size. In the present study we have examined interspecific and intraspecific variation in blood cell DNA levels in the genus Desmognathus, which shows greater variation in life history traits than any other salamander genus. Specimens of Desmognathus quadramaculatus, D. monticola, D. ochrophaeus and D. wrighti were collected from nature at two localities in the southern Appalachian Mountains. Estimates of genome size in pg of DNA were obtained from blood smears by DNA-Feulgen cytophotometry, using erythrocyte nuclei of Xenopus laevis as an internal reference standard of 6.35 pg DNA per cell. C-values of Desmognathus are the smallest in the order Caudata. Although significant variation in DNA levels was found among the four species, the differences were small, and do not support previously proposed relationships between C-value and life-history variation.

Improved in situ hybridization and G-banding by pretreatment with Denhardt's solution and gelatin-chrome alum.

Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity during in situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline-citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 X SSC for 2 min at 70 degrees C. A 3H-labelled, cloned DNA fragment of the highly repetitive AGM component alpha DNA was hybridized to the chromosomes by incubation at 45 degrees C for 16 h. Treatment with gelatin-chrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 X Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin after in situ hybridization.

Is there a limit on in situ, genomic replication in Drosophila?

The number of genomic replications of four separate tissues was determined in Drosophila melanogaster by summing the total number of nuclei and the maximum amount of DNA per nucleus in each tissue. It was found that for a given tissue the total number of nuclei was generally inversely proportional to the maximum amount of DNA per nucleus. In addition the sum of the number of divisions giving rise to the nuclei and the number of DNA replications occurring in the nucleus in each tissue never exceeded 20.

Cytophotometric evidence for the transformation of oocytes into nurse cells in Drosophila melanogaster.

A small proportion of ovarian chambers from females homozygous for the otu7 (for ovarian tumor) mutation contain an "oocyte" that in its nuclear morphology resembles a nurse cell. Such transformed oocytes also appear in colchicine-poisoned wild type ovaries. Cytophotometric estimates demonstrate that these oocytes have undergone 3-4 additional DNA replications, but that they lag behind the adjacent nurse cells by an average of 1.3 replication cycles. It follows that, under certain circumstances, the definitive oocyte can switch to the nurse cell developmental pathway and therefore that a mechanism normally exists for preventing the further replication of its DNA. In the case of otu7, oocytes sometimes restart their endocyclic DNA replications and produce paired, polytene, homologous chromosomes.

Determination of DNA content in the nurse and follicle cells from wild type and mutant Drosophila melanogaster by DNA-Feulgen cytophotometry.

DNA replication patterns in the nurse and follicle cells of wild type and a female sterile mutant, fs(1)1304, of Drosophila melanogaster have been studied by DNA-Feulgen cytophotometry, using a cell dispersal technique that allowed the measurement of DNA amounts in individual nuclei from egg chambers of known developmental stages. DNA-Feulgen values associated with various ovarian nuclei from egg chambers at different stages of development were used to assess a base line DNA content for ovarian tissues and to estimate the extent of DNA replication in the nurse cells and follicle cells of growing and mature egg chambers. Our data show that both the nurse and follicle cells undergo multiple cycles of endonuclear DNA replication and that there may be selective amplification as well as underreplication by portions of the genome in these highly polyploid, ovarian cells. Alternative models are proposed to account for the DNA replication patterns observed. Comparisons of DNA-Feulgen levels in wild type ovarian nuclei with those found for the fs(1)1304 mutant and its heterozygote in the balanced stock fs/FM3, show that equivalent DNA levels are present in follicle cell nuclei from all three types of females. Nurse cell nuclei in the homozygous fs stock, however, fail to achieve the same high DNA levels observed in both fs/FM3 and wild type nurse cell nuclei. Although the nuclei of follicle cells in ovaries from fs/fs females appear morphologically like those surrounding egg chambers in wild type ovaries, nurse cell nuclei from mutant females show a more compacted organization of their chromatin than found for nurse cell nuclei from wild type ovaries at similar developmental stages. Our findings suggest that a major effect of the fs(1)1304 mutation may be on the coiling behavior of chromatin and the conformation of DNA-protein moieties in both nurse cell and follicle cell nuclei. These changes in chromatin structure apparently are manifest by perturbations in DNA replication patterns and normal gene function in these biosynthetically active cells.

Cytophotometric comparisons of DNA levels in neuronal and glial cells of the cerebellum: a comparative study.

Several cytochemical studies of the DNA content and ploidy status of neuronal cell nuclei in the central nervous system have reported the occurrence of hyperdiploid amounts of DNA in Purkinje cells and suggest the existence of some type of 'extra' DNA, the biological significance of which is, as yet, unknown. To explore this phenomenon further, the DNA content of glial and Purkinje cell nuclei was determined in several vertebrate species, using the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) to stain isolated cerebellar nuclei for analysis with a single parameter flow cytometer. The Feulgen reaction for DNA was used to stain liver and cerebellar tissue imprints for the measurement of individual nuclei with a Vickers M86 integrating microdensitometer. In both types of analyses, chicken erythrocyte nuclei served as an internal reference standard of 2.5 pg DNA per cell. The mean DNA content of Purkinje cells and glial or granule cells was essentially the same as that found for diploid (2C) non-neuronal cells, such as hepatocytes, in rainbow trout, Amazon molly fish, salamander (Plethodon), mouse, rat, rabbit, cat, dog, monkey and human. Although Purkinje cell nuclei with 4C DNA levels were found in all of these species, except salamander and rabbit, the frequency of such cells was low (1-7%) and varied with the species. There was a low incidence of Purkinje cell nuclei with interclass DNA amounts in all species examined. Our data show that most neuronal cell nuclei in the cerebellum contain 2C levels of DNA.